Biosensor

ABSTRACT

In a biosensor comprising an electrically insulating base plate, an electrode system including a working electrode and a counter electrode formed on the base plate, and a reaction layer formed on or in the vicinity of the electrode system, at least surface of the reaction layer is made porous, so as to provide a biosensor of good response characteristic in which a reaction layer containing an enzyme dissolves quickly into a small amount of a sample solution and the enzyme reaction is effectively utilized.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a biosensor for determining thequantity of a substrate (specific component) contained in a sample,e.g., biological samples such as blood and urea, materials and productsin food industries and fruit juice, in a highly accurate, speedy andeasy manner.

[0002] There has been provided a biosensor capable of easily determiningthe quantity of a specific component (substrate) in biological samplesand foods without diluting or stirring the sample solution. For example,Japanese Laid-Open Patent Publication No. HEI 3-202764 discloses abiosensor including an electrode system formed on an electricallyinsulating base plate by screen printing and a reaction layer formed onthe electrode system. The reaction layer contains oxidoreductase and anelectron mediator (electron acceptor).

[0003] This biosensor determines the substrate concentration in a samplein the following manner.

[0004] First, a sample solution is dropped onto the reaction layer ofthe biosensor. Then, the reaction layer dissolves to cause an enzymereaction between the substrate in the sample solution and oxidoreductasein the reaction layer. Subsequently to the enzyme reaction, the electronmediator is reduced. After a predetermined time, a voltage is applied tothe electrode of the sensor to electrochemically oxidize the reducedelectron mediator. At that time, an oxidation current is obtained, fromwhich the substrate concentration in the sample solution is determined.

[0005] However, since the reaction layer of the prior art biosensor isformed by a method of dropping and drying a solution containing awater-soluble component, it is difficult to form the reaction layer intoa thin film. Further, it is also difficult to dry the reaction layeruniformly throughout its thickness and the performance of the sensor mayunfavorably vary depending on the dry state of the surface of thereaction layer. Accordingly, the enzyme needs to be contained in anexcessive amount in the reaction layer as compared with the amount ofthe substrate contained in the sample. Further, since the reagent iscontained in a large amount in the reaction layer, it takes long time todissolve the reaction layer.

[0006] Japanese Laid-Open Patent Publication No. 2001-208716 discloses asensor strip comprising an electrode support, an electrode set formedthereon and microscopic grains. In this sensor, the microscopic grainsare used to reduce the amount of the sample required for themeasurement, thereby increasing the dissolution rate of the reagent.

BRIEF SUMMARY OF THE INVENTION

[0007] In view of the-above described problems concerning the prior art,an object of the present invention is to provide a biosensor of goodresponse characteristic in which the reaction layer containing theenzyme dissolves rapidly into a small amount of a sample solution andthe enzyme reaction is effectively utilized.

[0008] Another object of the present invention is to provide a biosensorin which the above-described reaction layer is easily formed.

[0009] To achieve the above-described objects, the present inventionprovides a biosensor comprising an electrically insulating base plate,an electrode system including a working electrode and a counterelectrode formed on the base plate, and a reaction layer formed on or inthe vicinity of the electrode system, at least a surface of the reactionlayer being porous.

[0010] To make the reaction layer porous, it is effective that thereaction layer contains at least an enzyme and an aggregate of fineparticles having an average diameter of not smaller than 0.1 μm and notlarger than 1 μm.

[0011] The reaction layer may entirely be porous.

[0012] The porosity of the reaction layer is preferably 50% or more andthe upper limit thereof may be about 90%.

[0013] It is effective that the fine particles are made of a materialselected from the group consisting of a polymer compound, ceramic,glass, diamond and carbon.

[0014] It is also effective that the reaction layer further contains anelectron mediator.

[0015] A layer containing the electron mediator may be formed in-thevicinity of the reaction layer.

[0016] Further, it is effective that the reaction layer containscholesterol esterase, a surfactant and at least one of cholesteroloxidase and cholesterol dehydrogenase and that a layer containing theelectron mediator is formed in the vicinity of the reaction layer.

[0017] While the novel features of the invention are set forthparticularly in the appended claims, the invention, both as toorganization and content, will be better understood and appreciated,along with other objects and features thereof, from the followingdetailed description taken in conjunction with the drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0018]FIG. 1 is a perspective view of a disassembled biosensor accordingto an example of the present invention, in which a reaction layer isomitted.

[0019]FIG. 2 is a schematic vertical section of the above biosensor.

[0020]FIG. 3 is a graph illustrating comparison among responsecharacteristics of sensors according to Examples 1 and 2 and ComparativeExample 1.

[0021]FIG. 4 is an optical photomicrograph of the reaction layer formedin Example 1.

[0022]FIG. 5 is a graph illustrating comparison between responsecharacteristics of sensors of Example 5 and Comparative Example 3.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The biosensor of the present invention comprises an electricallyinsulating base plate, an electrode system including a working electrodeand a counter electrode formed on the base plate, and a reaction layerformed on or in the vicinity of the electrode system. The biosensor ischaracterized in that at least a surface of the reaction layer isporous.

[0024] To make the reaction layer porous, it is effective that thereaction layer contains at least an enzyme and an aggregate of fineparticles having an average diameter of not smaller than 0.1 82 m andnot larger than 1 μm.

[0025] The reaction layer may entirely be porous.

[0026] The fine particles used herein are preferably made of a materialselected from the group consisting of a polymer compound, ceramic suchas silica, alumina and titanium oxide, glass, diamond and carbon.

[0027] In the present invention, the reaction layer having at least aporous surface is preferably formed by dispersing the fine particles ina solution for forming the reaction layer, preferably an aqueoussolution containing an enzyme, spreading the resulting solution on or inthe vicinity of the electrode system on the base plate and drying thesolution.

[0028] If the average diameter of the fine particles is 1 μm or more,the fine particles do not disperse smoothly in the solution for formingthe reaction layer, which requires constant stirring. Thereby, theproduction process is complicated. Further, if the solution in which thefine particles are dispersed inhomogeneously is used, the resultingreaction layer becomes inhomogeneous and the porous reaction layercannot be obtained. In such a case, the resulting reaction layer doesnot dissolve quickly on a whole in use of the biosensor, whichdeteriorates the response characteristic.

[0029] On the other hand, if the average diameter of the fine particlesis smaller than 0.1 μm, voids in the fine particles in the resultingreaction layer are narrowed, the porosity of the reaction layerdecreases and the surface area of the enzyme does not increase.

[0030] Considering above, the present invention utilizes the fineparticles having the average diameter of not smaller than 0.1 μm and notlarger than 1 μm. Thereby, fine voids are formed in the reaction layerand hence the reaction layer becomes porous. This allows the samplesolution supplied to the sensor to permeate rapidly into the reactionlayer while dissolving the enzyme. As a result, the reaction layerquickly dissolves into the sample solution and the enzyme reactionproceeds speedily. Thus, highly accurate measurement is realized withoutcausing variations in response characteristics.

[0031] The amount of the fine particles contained in the reaction layeris suitably 10 to 99% by volume ratio with respect to the reaction layerwith a view to forming a thin enzyme layer on the fine particle surface.Thereby, the porous reaction layer which dissolves easily into thesample solution is formed. More preferably, the volume ratio is 10 to20%.

[0032] Examples of the enzyme used in the present invention includeglucose oxidase, glucose dehydrogenase, lactate oxygenase, lactatedehydrogenase, fructose oxidase, cholesterol oxidase, cholesteroldehydrogenase, cholesterol esterase, mutarotase, invertase, ascorbateoxidase, alcohol oxidase and the like. Among them, one or a combinationof two or more may be used.

[0033] The reaction layer of the biosensor according to the presentinvention may contain an electron mediator as described above. Examplesof the electron mediator include ferricyanide and salts thereof,p-benzoquinone and derivatives thereof, phenazine methosulfate,methylene blue, ferrocene and derivatives thereof and the like. Amongthem, one or a combination of two or more may be used as the electronmediator. If the electron mediator is not contained in the reactionlayer, the quantity of the substrate can be determined by using anenzyme or hydrogen peroxide in the sample solution.

[0034] If the electron mediator is mixed with the enzyme to form thereaction layer, the activity of the enzyme may be deteriorated by theelectron mediator during storage. Accordingly, it is preferred that theelectron mediator is contained in a layer isolated from the enzyme. Inthe case of a cholesterol sensor, are used a surfactant, cholesterolesterase for converting cholesterol ester into free cholesterol, and atleast one of cholesterol oxidase and cholesterol dehydrogenase. In sucha sensor, the electron mediator is preferably contained in a layerisolated from the enzymes.

[0035] A solvent for dissolving therein the enzyme and/or the electronmediator to prepare a solution for forming the reaction layer may beselected from water, various aqueous solutions such as phosphoric acidbuffer, Tris-HCl buffer, phosphoric acid buffer, acetic acid buffer andsodium chloride aqueous solution and organic solvents such as methanol,ethanol, toluene and acetone. The solvent is preferably one of them or amixture solution of two or more of them. The content of the fineparticles to be added to the solution for forming the reaction layer ispreferably in the range of 5 to 20% in terms of volume concentration.

[0036] The reaction layer of the biosensor according to the presentinvention may contain a hydrophilic polymer in addition to the enzymesand the electron mediator. The addition of the hydrophilic polymer tothe reaction layer prevents the reaction layer from peeling off the baseplate or the surface of the electrode system. Further, the fracture ofthe reaction layer surface is also prevented, which is effective inenhancing the reliability of the biosensor. The hydrophilic polymer maycover the electrode system, causing the same effect as the above.

[0037] Examples of the hydrophilic polymer include carboxymethylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxy methylethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acidsuch as polylysine, polystyrene sulfonate, gelatin and derivativesthereof, polymers of acrylic acid or derivatives thereof, polymers ofmaleic anhydride or salts thereof and starch or derivatives thereof.Among them, carboxymethyl cellulose is particularly preferred.

[0038] An oxidation current may be measured by a measurement method on atwo-electrode system using only a measuring electrode and a counterelectrode or a three-electrode system using a reference electrode inaddition, among which the three-electrode system allows measurement withgreater accuracy.

[0039] Hereinafter, a preferred embodiment of the biosensor according tothe present invention is described with reference to the figures.

[0040]FIG. 1 is a perspective view of a disassembled biosensor accordingto an embodiment of the present invention excluding a reaction layer.

[0041] Reference numeral 1 indicates an electrically insulating baseplate made of polyethylene terephthalate. On the base plate 1, leads 2and 3 made of silver paste are formed by screen printing. Further, bythe same printing method, are formed on the base plate 1 an electrodesystem including a working electrode 4 and a counter electrode 5 made ofconductive carbon paste containing a resin binder, and an electricallyinsulating layer 6 made of electrically insulating paste. Theelectrically insulating layer 6 exposes a certain area of the workingelectrode 4 and the counter electrode 5 and partially covers the leads.In other words, the electrically insulating layer 6 defines the exposedarea.

[0042] Other than silver and carbon, platinum, gold and palladium may beused as a material for the leads and the electrodes.

[0043] The biosensor is assembled from the insulating base plate 1 madeof polyethylene terephthalate, a cover 9, and a spacer 8 sandwichedbetween the base plate 1 and the cover 9. These components are bonded ina positional relationship as indicated by dashed lines shown in FIG. 1to form the biosensor. The spacer 8 is provided with a slit 10 forforming a sample solution supply pathway and the cover 9 includes an airaperture 11. With the base plate 1, the cover 9 and the spacer 8 bondedas shown in FIG. 1, a cavity is formed as a sample solution supplypathway between the base plate 1 and the cover 9. The cavity includes anopening end of the slit as a sample solution supply port and terminatesat the air aperture 11. In this explanation, the cover 9 and the spacer8 are combined as a covering member. However, a single member having agroove corresponding to the slit 10 of the spacer 8 may be used.

[0044] In FIG. 2, one or more reaction layers 7 containing at least anenzyme, an electron mediator and fine particles are formed on theinsulating base plate 1 provided with the electrode system as shown inFIG. 1, i.e., in the slit of the covering member. The reaction layer isnot necessarily formed on the electrode system as long as it is formedin a position exposed to the sample solution supply pathway and thesample solution flows over the electrode system while dissolving thereaction layer.

[0045] Hereinafter, the present invention is detailed by way ofexamples, but the invention is not limited thereto.

EXAMPLE 1

[0046] On the electrode system on the base plate 1 shown in FIG. 1, anaqueous solution containing fine particles of polystylene having anaverage diameter of 0.5 μm, potassium ferricyanide and glucose oxidase(EC1. 1. 3. 4, GOD) was dropped and dried in a warm-air dryer at 50° C.for 10 minutes to form a reaction layer 7. The obtained reaction layer 7contained 1 mg of the fine particles, 1 mg of potassium ferricyanide and0.05 mg of GOD, per cm².

[0047] Then, the base plate was combined with the spacer and the coverto complete the sensor and sensor characteristic as a glucose sensor wasevaluated.

[0048] On supplying a sample solution containing glucose to the reactionlayer 7, glucose in the sample solution was oxidized by GOD and at thesame time, the electron mediator in the reaction layer was reduced.After an elapse of a predetermined period of time, a voltage of +0.5 Vwith respect to the counter electrode 5 was applied to the workingelectrode 4 to oxidize the reduced electron mediator. Five secondslater, a current value between the working electrode and the counterelectrode was measured. The current value was proportional to theconcentration of the reduced electron mediator, i.e., the substrateconcentration in the sample solution. The glucose concentration in thesample solution was obtained from the current value.

[0049] Samples containing glucose in concentrations of 0 mg/dl, 180mg/dl, 360 mg/dl and 540 mg/dl were prepared and subjected to themeasurement for 30 seconds, respectively, to measure the responsecurrent value of the sensor with respect to each sample. As a result,the response current value and the glucose concentration had a certaincorrelation and showed favorable linearity. Also in the measurement for10 seconds, favorable linearity was obtained between the responsecurrent value and the glucose concentration. FIG. 3 shows the responsecharacteristic obtained in the measurement for 10 seconds, together withthe response characteristics obtained in Example 2 and ComparativeExample 1.

[0050] Further, with an optical microscope, the reaction layer 7 formedas described above was observed whether at least the surface was porousor not. FIG. 4 shows a photomicrograph taken under a magnification of42.5 times. As seen in FIG. 4, the surface of the reaction layer 7 wasporous.

[0051] The porosity of the reaction layer was obtained as a percentageof a volume of components in the reaction layer, which was obtained fromthe weight and specific gravity thereof, with respect to a volume of thereaction layer after drying. The porosity of the reaction layer wasabout 79.5%.

EXAMPLE 2

[0052] An aqueous solution containing potassium ferricyanide wasprepared, which was dropped and dried on the electrodes on the baseplate 1 shown in FIG. 1 to form a first layer. The content of potassiumferricyanide in the first layer was 1 mg per cm².

[0053] Then, an aqueous solution containing GOD and fine polymerparticles same as those of Example 1 was prepared, which was dropped onthe first layer and dried in a warm-air dryer at 50° C. for 10 minutesto form a second layer. The obtained second layer contained 1.0 mg ofthe fine polymer particles and 0.1 mg of GOD, per cm² of the secondlayer. The first and second layers were formed to function as thereaction layer 7.

[0054] In the same manner as Example 1, the response current value ofthe sensor with respect to a standardized glucose solution was measured.As a result, the response current value showed favorable linearity withrespect to the glucose concentration in both of the measurements for 30seconds and 10 seconds. The porosity of the reaction layer was about79%.

COMPARATIVE EXAMPLE 1

[0055] An aqueous solution containing potassium ferricyanide and GOD wasdropped on the electrodes on the base plate 1 shown in FIG. 1 and driedin a warm-air dryer at 50° C. for 10 minutes to form a reaction layer.The content of potassium ferricyanide in the reaction layer was 1 mg andthe content of GOD was 0.05 mg, per cm².

[0056] In the same manner as Example 1, the response current value ofthe sensor with respect to a standardized glucose solution was measured.As a result, the response current value and the glucose concentrationshowed favorable linearity in the measurement for 30 seconds. However,the linearity was not obtained with respect to glucose of highconcentration in the measurement for 10 seconds.

EXAMPLE 3

[0057] An aqueous solution containing fine particles of amorphoussilicon having an average diameter of 0.3 μm, potassium ferricyanide andGOD was prepared. Using the solution, the reaction layer 7 was formed onthe electrodes on the base plate 1 shown in FIG. 1 in the same manner asExample 1. The reaction layer 7 contained 1.5 mg of the fine particles,1 μg of potassium ferricyanide and 0.05 μg of GOD, per cm².

[0058] In the same manner as Example 1, the response current value ofthe sensor with respect to a standardized glucose solution was measured.As a result, the response current value showed favorable linearity withrespect to the glucose concentration in both of the measurements for 30seconds and 10 seconds. The porosity of the reaction layer was about74.5%.

EXAMPLE 4

[0059] An aqueous solution containing potassium ferricyanide wasprepared, which was dropped and dried on the electrodes on the baseplate 1 shown in FIG. 1 to form a first layer. The content of potassiumferricyanide in the first layer was 1 mg per cm².

[0060] Then, an aqueous solution containing GOD and fine particles ofamorphous silicon same as those in Example 3 was prepared, which wasdropped on the first layer and dried in a warm-air dryer at 50° C. for10 minutes to form a second layer. In the obtained second layer, thecontent of the fine polymer particles was 1.5 mg and the GOD content was0.1 mg, per cm². The first and second layers were formed to function asthe reaction layer 7.

[0061] In the same manner as Example 1, the response current value ofthe sensor with respect to a standardized glucose solution was measured.As a result, the response current value showed favorable linearity withrespect to the glucose concentration in both of the measurements for 30seconds and 10 seconds. The porosity of the reaction layer was about74%.

COMPARATIVE EXAMPLE 2

[0062] An aqueous solution containing potassium ferricyanide wasprepared, which was dropped and dried on the electrodes on the baseplate 1 shown in FIG. 1 to form a first layer. The content of potassiumferricyanide in the first layer was 1 mg per cm².

[0063] Then, an aqueous solution containing GOD and fine particles ofamorphous silicon having an average diameter of 1.5 am was prepared.However, in this aqueous solution, the fine particles were precipitatedimmediately and hence the solution required constant stirring during adropping step. This solution was dropped on the first layer and dried ina warm-air dryer at 50° C. for 10 minutes to form a second layer. Thatis, the first and second layers were formed to function as the reactionlayer 7. The content of the fine polymer particles in the thus formedsecond layer was 1.3 mg per cm² of the reaction layer. However, thecontent of the fine polymer particles was varied among sensors, whichcaused variations in GOD amount. The GOD amount was 0.1 mg on averageper cm² of the reaction layer.

[0064] In the same manner as Example 1, the response current value ofthe sensor with respect to a standardized glucose solution-was measured.The response current value was dependent on the glucose concentration inboth of the measurements for 30 seconds and 10 seconds. However, whenplural sensors were subjected to the measurement of the response currentvalue with respect to the same standardized glucose solution, theresponse current values were varied in a wide range.

EXAMPLE 5

[0065] A first layer containing potassium ferricyanide was formed in thesame manner as Example 2.

[0066] Then, an aqueous solution containing cholesterol esterase,cholesterol oxidase, fine particles of amorphous silica having anaverage diameter of 0.5 μm and Triton X-100 as a surfactant wasprepared. The solution was dropped on the first layer and dried in awarm-air dryer at 50° C. for 10 minutes to form a second layer. That is,the first and second layers were formed to function as the reactionlayer 7. The second layer contained 0.10 mg of cholesterol oxidase and0.05 mg of cholesterol esterase, per cm² of the reaction layer. Thecontent of the fine particles was 1 mg and that of Triton X100 was 0.4mg, per cm² of the reaction layer.

[0067] A cholesterol sensor was fabricated in the same manner as theproduction of the glucose sensor and the response current value of thesensor with respect to a sample solution containing cholesterol wasmeasured. As a result, the response current value showed favorablelinearity with respect to the cholesterol concentration in themeasurements for 3 minutes and 1 minute.

[0068]FIG. 5 shows the response characteristics of the sensors of thisexample and Comparative Example 3 in the measurement for 10 seconds. Theporosity of the above reaction layer was about 74.5%.

COMPARATIVE EXAMPLE 3

[0069] On the electrode system on the base plate 1 shown in FIG. 1, anaqueous solution mixture of potassium ferricyanide, cholesterol oxidaseand cholesterol esterase was dropped, which was dried in a warm-airdryer at 50° C. for 10 minutes to form a reaction layer 7. The obtainedreaction layer 7 contained 1 mg of potassium ferricyanide, 0.1 mg ofcholesterol oxidase and 0.05 mg of cholesterol esterase, per cm² of thereaction layer 7.

[0070] In the same manner as Example 3, the response current value ofthe sensor was measured. As a result, favorable linearity was notobtained in both of the measurements for 1 minute and 3 minutes.

COMPARATIVE EXAMPLE 4

[0071] A first layer containing potassium ferricyanide was formed in thesame manner as Example 3.

[0072] Then, an aqueous solution containing cholesterol esterase,cholesterol oxidase, fine particles of amorphous silicon having anaverage diameter of 1.5 am and Triton X-100 as a surfactant wasprepared. The solution was dropped on the first layer and dried in awarm-air dryer at 50° C. for 10 minutes to form a second layer. That is,the first and second layers were formed to function as the reactionlayer 7. In this solution, however, the fine particles were immediatelyprecipitated, which required constant stirring during the dropping step.The second layer contained 0.10 mg of cholesterol oxidase and 0.05 mg ofcholesterol esterase per cm² of the reaction layer. The content of thefine particles was 0.5 to 1.5 mg and that of Triton X-100 was 1.0 mg,per cm² of the reaction layer.

[0073] In the same manner as Example 3, the response current value ofthe sensor was measured. As a result, favorable response linearity wasnot obtained in both of the measurements for 1 minute and 3 minutes. Thereason is presumably that the fine particles in the solution for formingthe reaction layer were hard to disperse homogeneously and the fineparticle amount in the reaction layer assembled in the sensor was notuniform, which caused considerable variations in response value of thesensor in evaluating the response characteristics.

EXAMPLE 6

[0074] On a platinum electrode system formed on the base plate 1 shownin FIG. 1, an aqueous solution containing glucose oxidase (GOD) and fineparticles (beads) of polystylene having an average diameter of 0.8 μmwas dropped to form a reaction layer 7. The obtained reaction layer 7contained 0.03 mg of GOD and 1.5 mg of the fine particles, per cm² ofthe reaction layer.

[0075] On the electrode system, a sample solution containing glucose wassupplied. Glucose in the sample solution was oxidized by GOD to generatehydrogen peroxide. Ten seconds after the sample solution was supplied, avoltage of +1.0V with respect to the counter electrode 5 was applied tothe working electrode 4 to measure a current value after 5 seconds. Thiscurrent value was proportional to the substrate concentration in thesample solution, from which the glucose concentration in the samplesolution was obtained.

[0076] Samples containing glucose in concentrations of 0 mg/dl, 180mg/dl, 360 mg/dl and 540 mg/dl were prepared and subjected to themeasurement for 30 seconds, respectively, to measure the responsecurrent value of the sensor with respect to each solution. As a result,the response current value and the glucose concentration had a certaincorrelation and showed favorable linearity. Further, also in themeasurement for 10 seconds, favorable linearity was obtained between theresponse current value and the glucose concentration. The porosity ofthe reaction layer was about 84.7%.

[0077] In the case where the fine particles were not mixed in thereaction layer, the response linearity was lost with respect to thesubstrate of high concentration. The reason for the above is supposedlythat the reaction layer without the fine particles is dried to have adense structure, and hence only the surface is dissolved to causereaction with the sample solution supplied to the sensor.

[0078] As compared with the reaction layer without the fine particles,in the reaction layer formed by mixing the fine particles the enzyme andthe electron mediator are dried into a thin film on the fine particlesurface. Since the sample solution permeates into the reaction layervery rapidly through the voids in the fine particles, the reagentincluding the enzyme dissolves smoothly and the enzyme reaction proceedspromptly. As a result, the measurement with the sensor can be carriedout in a reduced period.

[0079] In this case, if the fine particles contained in the reactionlayer have an average diameter smaller than 0.1 μm, the structure of thereaction layer containing the fine particles becomes very minuscule andvoids are not generated in the fine particles. Therefore, the reactionlayer does not dissolve smoothly into the sample solution. On the otherhand, if the average diameter of the fine particles is 1 μm or more, thefine particles may precipitate in the solution for forming the reactionlayer or coagulate on or near the solution surface and hence theproduction process is complicated.

[0080] From the above, the effective size of the fine particles is inthe range of not smaller than 0.1 μm and not larger than 1 μm. With thereaction layer containing the fine particles so defined, the responsecharacteristic of the sensor becomes very favorable even in measuring asample solution containing various components such as hemocytes andprotein, e.g., blood.

[0081] In the above-described examples, explanation is given in terms ofthe glucose sensor and the cholesterol sensor. However, the presentinvention is also applicable to other sensors such as a glucose orcholesterol sensor using other enzymes, a lactic acid sensor, a fructosesensor, a sucrose sensor, an alcohol sensor, an ascorbic acid sensor andthe like.

[0082] As described above, according to the present invention, there isprovided a biosensor capable of determining the quantity of a substratecontained in a sample, e.g., biological samples such as blood and ureaand materials and products in food industries, in a highly accurate,speedy and easy manner.

[0083] Although the present invention has been described in terms of thepresently preferred embodiments, it is to be understood that suchdisclosure is not to be interpreted as limiting. Various alterations andmodifications will no doubt become apparent to those skilled in the artto which the present invention pertains, after having read the abovedisclosure. Accordingly, it is intended that the appended claims beinterpreted as covering all alterations and modifications as fall withinthe true spirit and scope of the invention.

1. A biosensor comprising an electrically insulating base plate, anelectrode system including a working electrode and a counter electrodeformed on the base plate, and a reaction layer formed on or in thevicinity of the electrode system, at least a surface of the reactionlayer being porous.
 2. The biosensor according to claim 1, wherein thereaction layer contains at least an enzyme and an aggregate of fineparticles having an average diameter of not smaller than 0.1 μm and notlarger than 1 μm.
 3. The biosensor according to claim 1, wherein thefine particles are made of a material selected from the group consistingof a polymer compound, ceramic, glass, diamond and carbon.
 4. Thebiosensor according to claim 1, wherein the reaction layer furthercontains an electron mediator.
 5. The biosensor according to claim 1,wherein a layer containing an electron mediator is formed in thevicinity of the reaction layer.
 6. The biosensor according to claim 1,wherein the reaction layer contains cholesterol esterase, a surfactantand at least one of cholesterol oxidase and cholesterol dehydrogenase,and a layer containing an electron mediator is formed in the vicinity ofthe reaction layer.